Classifications

• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
  • C03C17/34—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
  • C03C17/42—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions at least one coating of an organic material and at least one non-metal coating
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
  • C03C17/006—Surface treatment of glass, not in the form of fibres or filaments, by coating with materials of composite character
  • C03C17/007—Surface treatment of glass, not in the form of fibres or filaments, by coating with materials of composite character containing a dispersed phase, e.g. particles, fibres or flakes, in a continuous phase
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
  • C03C17/02—Surface treatment of glass, not in the form of fibres or filaments, by coating with glass
  • C03C17/04—Surface treatment of glass, not in the form of fibres or filaments, by coating with glass by fritting glass powder
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C8/00—Enamels; Glazes; Fusion seal compositions being frit compositions having non-frit additions
  • C03C8/14—Glass frit mixtures having non-frit additions, e.g. opacifiers, colorants, mill-additions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
  • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00659—Two-dimensional arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/00722—Nucleotides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/00725—Peptides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/0074—Biological products
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/0074—Biological products
  • B01J2219/00743—Cells
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C2217/00—Coatings on glass
  • C03C2217/40—Coatings comprising at least one inhomogeneous layer
  • C03C2217/425—Coatings comprising at least one inhomogeneous layer consisting of a porous layer
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C2218/00—Methods for coating glass
  • C03C2218/10—Deposition methods
  • C03C2218/11—Deposition methods from solutions or suspensions
  • C03C2218/114—Deposition methods from solutions or suspensions by brushing, pouring or doctorblading
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C2218/00—Methods for coating glass
  • C03C2218/10—Deposition methods
  • C03C2218/11—Deposition methods from solutions or suspensions
  • C03C2218/119—Deposition methods from solutions or suspensions by printing
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

• the present invention relates to a glass material that forms at least a part of a porous substrate used for biological or biochemical assays.
• the invention pertains to material compositions that exhibit reduced levels of auto-fluorescence under certain light wavelengths.
• microarray technology has blossomed.
• Biological, pharmaceutical, and other research communities have recognized that microarrays are useful, high-tliroughput research tools to measure a variety of biological or biochemical functions.
• the microarray format is likely to remain a key research tool into the foreseeable future.
• Applications for microarray technology will continue to expand in the areas of drug discovery and development, diagnostic assays, and biological research.
• Bio or chemical probe molecules can be immobilized on a solid surface for many kinds of assays.
• high-density arrays have become invaluable tools for drug researchers and geneticists in a variety of binding assays, such as to obtain information on the expression of genes.
• SNP single nucleic acid polymorphism
• Clinical and research laboratories are increasingly using DNA testing as a means to determine genetic risk factors for diseases like breast cancer, heart disease, Alzheimer's disease, etc.
• a high-density array typically comprises between 2,000 and 50,000 probes, with the possibility of up to about 80,000 or 100,000 probes, in the form of single stranded DNA, each of a known and different sequence, arranged in a predetermined pattern on a substrate.
• DNA arrays can be used to study the regulatory activity of genes, wherein certain genes are turned on or "up-regulated” and other genes are turned off or "down-regulated.” So, for example, a researcher can compare a normal colon cell with a malignant colon cell and thereby determine which genes are being expressed or not expressed in the aberrant cell.
• the regulatory sites of genes serves as key targets for drug therapy.
• DNA arrays have for years been printed onto organic, micro-porous membranes such as nylon or nitrocellulose.
• the densities at which one can print DNA solutions onto these types of organic micro-porous membranes is limited because of the tendency for the DNA solution to wick laterally through the membrane, thus causing cross-talk and contamination between adjacent locations.
• Others have employed a flat, non- porous substrate surface made from glass. (See for example, U.S. Patent No. 5,744,305, incorporated herein by reference.)
• Non-porous, planar, solid substrates, such as microscope slides, coated with a functionalized layer have been a preferred surface upon which to deposit or print a variety of probe molecules for microarrays.
• a typical porous substrate comprises a base substrate that is either micro-porous or non-porous, and having either pores formed therein, or a porous layer bonded to a top surface of the base substrate.
• the base substrate is preferably made from a suitable glass.
• porous glass substrates can generate high signal intensity, they unfortunately also have an increased level of background auto-fluorescence or noise, which can be detrimental to the overall signal to noise ratio.
• background fluorescence from the substrate's surface under certain light wavelengths can obscure or optically "wash out” the signal emitted from fluorescently labeled binding partners of immobilized probe molecules.
• a high level of auto-fluorescence in the substrate prevents the user from accurately detennining a baseline level of fluorescence. Hence, assay detection and analysis may suffer.
• Previous attempts to reduce auto-fluorescence by means other than the addition of colorants were unsuccessful.
• the auto- fluorescence background signal was reduced but the porous structure, unfortunately, also was affected.
• the porous layer glass had fused, losing porosity, hence the H 2 -N 2 - fired slide had little structural difference from a conventional, two-dimensional flat slide surface.
• the present invention addresses the issue of high auto-fluorescence or reflectivity in porous substrates without suffering the adverse effects of previous techniques.
• a glass frit composition was modified to reduce reflectance in an attempt to decrease the level of intrinsic auto-fluorescence of microarray substrates.
• Colorant ions were incorporated into the glass composition in order to absorb and reduce stray reflected light, but without seriously reducing the overall desirable fluorescence signal.
• cobalt or nickel (II) oxides separately or in a combination with each other or other transition metal species added to a porous glass composition, one can create a tint that reduces reflectance and background signal due to scattering.
• the net signal for Cy5 and Cy3 labels are both respectively lower with a tinted porous substrates by at least twenty or twenty-five percent.
• Signal to noise ratio for a tinted porous substrate is dramatically improved over white porous glass or inorganic materials.
• the reduction in Cy5 generated background can significantly impact the signal to noise metric for microarray analysis.
• the present invention in one aspect, includes a porous substrate having: a support; and a porous region on a surface of said support.
• the porous region is composed of a primarily inorganic material and having a surface upon which a number of probe molecules can be immobilized.
• the porous region also has a tint and exhibits a reduction in relative reflectance and auto-fluorescence levels by at least about 15% or 20%, preferably about 50%, over a non-tinted porous substrate surface, over a wavelength range from about 400 or 420 nm to about 700 or 720 nm.
• the tinted porous region has a colorant component, including a transition metal ion, incorporated into its composition.
• the tinted porous region may have a composition in weight percent consisting essentially of: 53-67% SiO 2 ; 3-10% Al 2 O 3 ; 12-24% B 2 O 3 ; 0-5% K 2 O; 0-2% MgO; 0.5-3% CaO; 0-3% SrO; 2-7% BaO; 0-2% Sb 2 O 3 , and at least one of the following, either individually or in combination, 0.1-9% Co 3 O ; 0.1-10% NiO; or 0- 10% R ⁇ O y , wherein R is a transition metal, and x and y are each >0.
• the R transition metals may include Fe, V, or Cu.
• the porous layer has a composition that consists essentially of: 55-65% SiO 2 ; 4-9% Al 2 O 3 ; 14-21% B 2 O 3 ; 1-5% K 2 O; 0.1- 2% MgO; 1-2.5% CaO; 0.5-1.75% SrO; 3-5% BaO; 0-2% Sb 2 O 3 , and at least one of the following, either individually or in combination, 0.1-8% Co 3 O 4 ; 0.1-10% NiO; 0-10% R ⁇ O y , preferably with the cobalt and nickel species both present.
• the glass compositions are chemically and mechanically durable, and have a coefficient of thermal expansion (CTE) of between about 35-44 x 10 "7 /°C, preferably 38-40 x 10 "7 /°C, which is suitable to bind securely with typical non-porous glass slides or substrates.
• CTE coefficient of thermal expansion
• the porous substrate is prepared with a number of biological or chemical probes.
• the probe species or molecules are attached at defined locations on or within the tinted porous layer.
• a set of probes at defined locations form a microarray of probe microspots having a density of at least one microspot per cm 2 , preferably at least 10 microspots per cm 2 , more preferably about 20- 100 microspots or more per cm 2 .
• FlG. 1 is a graphical representation comparing the relative average background fluorescence of an uncoated a tinted porous glass layer on a substrate (I) according to the present invention with a control (A), an un-tinted "white” porous layer (B), and a flat non-porous substrate surface (C), each of which are also uncoated. Table 1 summarizes the data of this graph.
• FlG. 2 is a graphical representation comparing the relative mean local background auto-fluorescence of a tinted porous glass layer on a substrate with a control (A), an un-tinted "white” porous layer (B), and a flat non-porous substrate (C), each of which is coated with a layer of 5% gamma-amiiiopropylsilane (GAPS) and treated with a reducing agent, such as NaBH .
• GAPS gamma-amiiiopropylsilane
• FlGs. 3 A and 3B are graphical representations of the net fluorescence signals (i.e., mean signal less mean background) for cyanine dyes, Cy5 and Cy3, respectively.
• FlGs. 4A, 4B, and 4C are scanned false-color images of three substrates. Serving as a control for comparison purposes, Fig. 4A is an image of a flat plane of a non-porous glass, such as used in flat-panel display or LCD devices.
• Fig. 4B is an image of a tinted porous glass surface, according to the present invention.
• Fig. 4C is an image of a prior "white" porous glass surface.
• FlGs. 5A and 5B are false color images of arrays that have undergone hybridization, and show, in Cy5-channel wavelength, a comparison between tinted and un-tinted porous layers, respectively, on slides that were not treated with a pre- hybridization buffer containing NaBH .
• FlGs. 6A and 6B are false color images of arrays that have undergone hybridization, and show, in Cy3-channel wavelength, a comparison between tinted and un-tinted porous layers, respectively, on slides that were treated with a pre- hybridization buffer containing NaBH .
• FIG. 7 is a graph comparing the signal to noise metric for samples of the different types of substrates.
• the tinted substrate exhibited the highest relative Cy5 signal of any of the substrate types.
• the signal-to-noise ratio for the tinted porous substrate, untreated with NaBH was at least 5 fold that of the corresponding untreated "white” porous surface, while the signal-to-noise ratio for the treated tinted substrate is about two times greater than the treated white surface.
• biological molecule refers to any kind of biological entity, including, such as, oligonucleotides, DNA, RNA, peptide nucleic acid (PNA), peptides, polypeptides, protein domains, proteins, fusion proteins, antibodies, membrane proteins, lipids, lipid membranes, cellular membranes, cell lysates, oligosaccharides, or polysaccharides, or lectins.
• PNA peptide nucleic acid
• biospot or “microspot” refers to a discrete or defined area, locus, or spot on the surface of a substrate, containing a deposit of biological or chemical material.
• complement refers to the reciprocal or corresponding moiety of a molecule to another.
• receptor-ligand pairs or complementary nucleic acid sequences, in which nucleotides on opposite strands that would normally base pair with each other according to Watson-Crick-base pair (A/T, G/C, C/G, T/A) correspondence.
• fluid or "film of fluid” as used herein refers to a material or medium that can flow such as a gas, a liquid, or a semisolid.
• the term "functionalization” as used herein relates to modification of a solid substrate to provide a plurality of functional groups on the substrate surface.
• the phrase “functionalized surface” as used herein refers to a substrate surface that has been modified to have a plurality of functional groups present thereon.
• the terms “nucleoside” and “nucleotide” are intended to include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purities or pyrimidines, or other heterocycles.
• nucleoside and nucleotide include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.
• amino acid is intended to include not only the L-, D- and noiichiral forms of naturally occurring amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), but also modified amino acids, amino acid analogs, and other chemical compounds which can be incorporated in conventional oligopeptide synthesis, e.g., 4-nitrophenylalanine, isoglutamic acid, isogmtamine, e-nicotinoyl-lysine, isonipecotic acid, tetrahydroisoquinoleic acid, ⁇ -aminoisobutyric acid, sarcosine, citrulline, cysteic acid, t-
• probe refers to either a natural or synthetic molecule, which according to the nomenclature recommended by B. Phimister (Nature Genetics 1999, 21 supplement, pp. 1-60.), is immobilized to a substrate surface.
• the corresponding microspots are referred to as “probe microspots,” and these microspots are arranged in a spatially addressable manner to form a microarray.
• molecules in the sample selectively and specifically binds to their binding partners (i.e., probes) in the microarrays.
• receptor refers to a molecule that has an affinity for a ligand. Receptors may be naturally-occurring or man-made molecules. They may be employed in their unaltered state or as aggregates with other species.
• receptors which may be employed according to this invention may include, but are not limited to, antibodies, monoclonal antibodies and antisera reactive with specific antigenic determinants, pharamaceutical or toxin molecules, oligonucleotides, polynucleotides, DNA, RNA, peptide nucleic acid (PNA), peptides, polypeptides, protein domains, proteins, fusion proteins, cofactors, lectins, oligosacharides, polysacharides, viruses, cells, cellular membranes, cell membrane receptors, and organelles. Receptors are sometimes referred to in the art as anti-ligands.
• a “ligand-receptor pair” is formed when two molecules have combined through molecular recognition to form a complex.
• sample as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, containing one or more components of interest.
• substrate refers to a solid or semi-solid material that is porous or semi-porous, which can form a stable support for immobilized probe molecules.
• substrate surface can be selected from a variety of materials.
• the materials may be biological (e.g., plant cell walls), non-biological, organic (e.g., silanes, polylycine, hydrogels), inorganic (e.g., glass, ceramics, SiO 2 , gold or platinum, or gold- or platinum-coated), polymeric (e.g., polyethylene, polystyrene, polyvinyl, polyester, etc.), or a combination of any of these, in the form of a slide, plate, film, particles, beads or spheres.
• the substrate surface is two dimensional and relatively flat, and fully porous for the printing of an array of biospots, but may take on alternative surface configurations.
• the substrate may be textured with raised or depressed regions.
• the substrate surface will have thereon at least one kind of functional or reactive group, which could be amino, carboxyl, hydroxyl, thiol groups, amine-reactive groups, thiol-reactive groups, Ni-chelating groups, anti-His-antibody groups, or the like.
• functional or reactive group which could be amino, carboxyl, hydroxyl, thiol groups, amine-reactive groups, thiol-reactive groups, Ni-chelating groups, anti-His-antibody groups, or the like.
• target(s),” “target moieties,” “target analyte,” “biological target,” or “chemical target” refers to a solvated particle, molecule or compound of interest in a sample that is to be detected and identified. Suitable targets include organic and inorganic molecules, biomolecules.
• the target may be an environmental pollutant (e.g., such as pesticides, insecticides, toxins, etc); a chemical (e.g., solvents, polymers, organic materials, etc); a therapeutic molecule (e.g., therapeutic and abuse drugs, antibiotics, etc); abiomolecule (e.g., hormones, cytokines, proteins, peptides, protein domains, fusion proteins, nucleotides, oligonucleotides, DNA, RNA, peptide nucleotide acids (PNA), genomic DNA, lipids, lipid membranes, carbohydrates, cellular membrane antigens, receptors or their ligands, etc); whole cells (e.g., pathogenic bacteria, eukaryotic cells, etc); a virus; or spores, etc.
• an environmental pollutant e.g., such as pesticides, insecticides, toxins, etc
• a chemical e.g., solvents, polymers, organic materials, etc
• Porous substrates like those described in U.S. Patent Publication Nos. 2003- 0003474, or 2002-0142339, or PCT Publication No. WO 00/61282, or an article by M. Glazer et ah, Colloidal Silica Films for High-Capacity DNA Probe Assays, Chem. Mater. 2001, 13, 4773-4782, the contents of each are incorporated herein by reference, have been in recent years a subject area of activity and development in the solid-phase biological assay field.
• a porous substrate involves a substantially flat, porous, inorganic layer applied or adhered to a non-porous support.
• a beneficial characteristic of porous substrates is their enhanced ability to retain nucleic and/or other probe moieties for high-density arrays.
• the porous surface provides a greater amount of surface area for immobilizing DNA probe molecules, for instance, which increases the density of nucleic acid binding sites per unit cross-sectional area of the substrate. The increased number of possible binding sites per unit area results in greater retention of immobilized nucleotide probes and the emission of an higher signal level when hybridized with target molecules.
• a porous inorganic surface that is properly treated with a coating of a binding agent, such as a poly-silane or cationic polymer, can also prevent lateral cross-talk.
• Porous ceramic or glass substrates for DNA-binding can consistently yield improved performance relative to both other porous and non-porous flat substrates, and can satisfy other requirements such as chemical and mechanical durability.
• the porous surfaces are fabricated by means of a tape-casting or a screen-printing process using respectively a ceramic or glass containing slip or paste/ink. Adjustments in firing temperature, firing time, and size of the ceramic or glass particles can control the size of the microstructures; hence, the porous layer may have a porosity ranging from about zero or one or two percent up to potentially 99%. Preferable porosity may range from about 55% to about 80% or 90%.
• Tape-cast porous borosilicate glass (Corning Inc., Code 7761) layers on calcium aluminosilicate glass slides (Corning Inc., Code 1737) tend to retain the greatest absolute quantity of nucleotides after printing and through all washing, blocking, hybridizing, and rinsing steps.
• Printed DNA bound on porous tape-cast borosilicate are accessible for hybridization, and exhibit higher absolute signals and signal-to-noise ratio than o achieved for porous glass slides or sol-gel coated slides.
• the performance of an HDA depends on several factors, such as composition and purity of the substrate, surface chemistry applied to the substrate, and quality of biological molecules applied at all stages of manufacture and use.
• a microarray is a sensor, and its response can be benchmarked using standard criteria.
• Three reference points of merit for any sensor are detection threshold, sensitivity, and dynamic range.
• the detection threshold is the level at which the smallest input to the sensor can be detected in the output response.
• the sensitivity relates the input signal to the output signal of the sensor in the dynamic range.
• the dynamic range in combination with the detection threshold, defines an upper limit for the response of the device. Inputs greater than some threshold value do not change the sensor output.
• a superior microarray is one with the lowest detection threshold, highest sensitivity, and widest dynamic range.
• the benefits of lower detection threshold are immediately apparent.
• Differential expression can be measured for genes expressed at lower concentrations of biological molecules.
• the accuracy of measurement in testing for differential expression is affected by sensitivity. Higher sensitivity provides greater accuracy, especially at concentrations near the detection threshold. With the higher sensitivity, uncertainty or error in intensity due to factors associated with excitation laser and photomultiplier detector in the scanner can be reduced. Thus, discrimination between smaller concentrations can be made with greater accuracy in differential gene expression.
• porous, inorganic substrates can provide significant advantages over prior inorganic and organic substrates for high-density DNA arrays.
• Porous inorganic substrates for arrays have superior sensitivity and lower detection threshold when compared to flat, nonporous surfaces.
• Porous inorganic substrates having certain types of microstructure can produce fluorescent-molecule sensitivities of one or more than two orders of magnitude greater than that of a flat, non-porous slide. Sensitivity is an important property for biological applications where detection of fluorescent molecules is required.
• a substrate with higher sensitivity is attractive for these applications since smaller changes in concentration and possibly lower overall concentrations can be more easily detected.
• Enhanced sensitivity and lower detection thresholds provide opportunities to reduce cost for the array manufacturer or user. Less material could be printed during manufacture, or the concentration of probes in hybridization solution could be reduced while still maintaining the same level of performance, if not a higher level than that of a flat slide.
• porous substrates Unfortunately exhibit a higher background or auto-fluorescence at certain wavelengths, particular in the visible light spectrum, between about 400 nm to 700 nm, than coated non-porous flat glass substrates. In an effort to overcome this problem, while preserving the beneficial aspects of porous substrates, we have developed the present invention.
• Auto-fluorescence or intrinsic background of porous glass materials can be reduced effectively, according to the present invention, by doping the glass composition with certain inorganic components to tint the porous glass layer. This would be more attractive to users of porous substrates who want a low background with high signal intensity.
• the present invention can both enhance sensitivity and improve threshold detection of fluorescence markers.
• a colorant incorporated into the composition of the porous inorganic component or coating layer on a porous substrate can reduce the relative level of reflectance and auto-fluorescence.
• cobalt oxide or nickel (II) oxide components which are used in Black Light Blue glass for conventional lighting applications, were incorporated into a glass composition to achieve a tint.
• the dark glass frit appears as a grayish layer after its is applied and fired to bond to the underlying non-porous backing or support.
• the colorant tint according to the invention consistently reduces background issues due to uncontrolled light scattering in a coated or bare porous glass layer.
• Table 3 presents possible ranges, according to some embodiments, for the major components of the tinted porous glass.
• Table 3 . _ wt% ' Lower range Upper range Si0 2 53 67 A1 2 0 3 5 10 B 2 0 3 12 24 ⁇ 2 o 0 5 MgO 0 1 CaO 1 3 SrO 0 3 BaO 3 7 Sb 2 0 3 0 2 Co 3 0 4 0 2 NiO 0 4 other Transition 0 4 Metals
• the preferred composition would still have cobalt and nickel oxides, but the other transition metal elements such as Fe, V or Cu, which exhibited little or no detectable increase (as 1 x 3 in slides) in auto-fluorescence can be included as well, although they are not as strongly absorbent of auto-fluorescence as Co and Ni. We observed additional fluorescence from Cr and Mil. Hence, one may exclude these elements. Also, it is possible to make these glasses without the Sb fining agent, and it might even be desirable to do that under some circumstances. [0044] In the working examples, borosilicate glass was selected as the porous layer since borosilicates are transparent and are readily available, although other glasses having similar physical characteristics may be substituted.
• the glass transition/sintering temperature of the substrate and porous layer should be similar so as to provide for strong adhesion between the two. Also, in the ideal situation, the surface is positively charged in a neutral aqueous solution, so as to aid in attaching the negatively charged DNA molecules.
• Table 4 provides a comparison between an example of an initial or original "white” borosilicate composition and a similar example according to the invention containing colorant components.
• Table 4 wt% Ex. Initial Ex, . 7 Base tinted SiO 2 64.16 63.35 A1 2 0 3 7.54 7.5 B 2 0 3 17.84 17.76 K 2 0 2.68 2.68 MgO 0.35 0.35 CaO 1.51 1.5 SrO 0.92 0.92 BaO 4.1 3.6 Sb 2 0 3 0.9 0.9 Co 3 0 4 0.4 NiO 1 other Transition 0.04 Metals 100 100
• Crushed borosilicate glass particles are sieved and wet-milled to a reduced particle size (average size in the range of about 0.07-3.5 ⁇ m).
• the particles were ball- milled for 24-72 hours using a one gallon bottle (Nalgene) charged with the crushed borosilicate glass, ZrO 2 milling cylinders and filled with isopropanol to about 85 percent full. After milling, the slurry was stirred and then allowed to stand without disturbance for the particles to settle. Settling can further control the size distribution of the glass particles before a binder is added.
• the liquid slurry was poured from the Nalgene bottle and the isopropanol was evaporated on a hot plate to recover the glass powder. Care was taken no to disturb the sediment at the bottom of the bottle.
• the average particle size of the borosilicate powder obtained after settling was in the range of about 0.05-1.5 ⁇ m.
• the borosilicate powder was used in preparation of slip for tape casting.
• U.S. Patent No. 5,089,455 incorporated herein by reference, describes in detail the preparation of zirconia based slips for the tape casting of thin zirconia electrolytes such as for fuel cell applications.
• Preparation of the borosilicate slip for casting of a porous layer was performed in analogous fashion according to the procedure given in that patent. The recipe was adjusted to account for the difference in density of ZrO 2 and borosilicate, and no settling was performed to narrow the particle size distribution.
• milled borosilicate powder 90.9 g ethanol, 21.98 g 1-butanol, 5.0 g propylene glycol, 6.25 g distilled water, 2.5 g Emphos, and 1125 g of one cm ZrO milling balls were weighed into a 500 ml nalgene bottle and vibratory milled for 72 hours.
• the milled slip was poured from the Nalgene bottle without the milling media into a new 250 mL Nalgene bottle.
• the final step in the preparation of the slip was to add 5.0 g of a 50 w/o mixture of glacial acetic acid and isopropanol, 8.75 g dibutylphthalate, and 15 g polyvinylbutyral, and five or six 1 cm zirconia milling balls.
• the bottle was then rolled gently at less than 1 rotation per second to thoroughly mix and remove bubbles for at least 72 hours prior to tape casting.
• Tape casting of the slips to form the porous substrates for microarrays is relatively straightforward. Using a non-porous understructure made from a calcium aluminosilicate glass (Corning Inc., Code 1737), a panel of glass scored to give 1 inch by 3 inch microscope slides was cleaned on both major surfaces.
• CTE coefficient of thermal expansion
• the coating should be allowed to dry before proceeding.
• the frit glass slip is cast on top using another tape casting blade. The coated slides were allowed to dry.
• the actual thickness of the porous layer is not necessarily limiting, in some embodiments.
• the dried and fired porous layers may be as thick as about 1 mm.
• the final porous layer in preferred examples range from about 5 or 6 ⁇ m to about 100 or 150 ⁇ m. More preferably, the thicknesses may range from about 10 to 75 ⁇ m or about 15 to 50 ⁇ m. A most desired fired thickness for the porous layer is about 30 ⁇ m ⁇ 5 ⁇ m.
• the 1737-glass panel can be snapped into individual slides and fired. These tape-cast slides were fired on alumina fiber board using an alumina fiber board cover. The coated slides were fired at a temperature that causes the bonding layer to fuse, and the top frit glass to sinter into a porous layer.
• the exact temperature (e.g., ⁇ 650-735°C) and duration (e.g., -2-3 hours) of firing may vary according to the glass composition or desired characteristics of the porous layer, hi general, porosity of the coatings decreases with increasing firing temperature.
• the substrates are allowed to cool to ambient temperature for 4 hours.
• the example slides along with a sample each of the original white porous slides and a plain uncoated 1737-glass LCD panel were fired at about 705°C, and were scanned after firing to determine the background.
• Fired slides are translucent and have a hazy appearance due to light scattering.
• the tinted porous layers have a grayish color.
• the porous layer should be strongly bonded to the calcium aluminosilicate glass substrate. Larger pores were -5 ⁇ m, and the smaller pores have an average size of -0.5 ⁇ m to -1.0 ⁇ m.
• This light scattering effect may in part be due to the microstructure features of the porous layer such as layer thickness, particle size, particle shape, pore size, pore shape, porosity, continuity of the glass and pore phases, surface density of binding sites, etc. Adjustments of these parameters may optimize the light scattering effect. Thus, a higher rate of light emission from the fluorescent molecules is possible in the porous layer provided that the two-level fluorescent system is not itself saturated.
• the light scattering effect and enhanced sensitivity disappear on infiltration of the pores of the coating with an index matching fluid such as glycerol.
• the superior sensor characteristics of a porous slide of the present invention are due to a higher surface area for binding of biological molecules, improved excitation of fluorophore due to scattering of excitation through the porous surface, and rapid hybridization kinetics.
• the porous surface can have greater density of binding sites per unit area for DNA attachment than a comparable flat nonporous substrate.
• An increase in the absolute number of retained DNA is important, since it minimizes the loss of DNA during the processing steps.
• the optical signal from the fluorescent tags on both the printed, known DNA strands and any hybridized, unknown strands is strengthened.
• the effective number of binding sites on the substrate increases with decreasing particle size and increasing thickness of the porous layer.
• Retention of DNA can be enhanced by the microstructural characteristics of a porous, nucleic-acid-binding surface.
• retention of printed DNA through washing, blocking, hybridizing, and rinsing operations is critical. Excessive loss of the printed DNA leads to a low fluorescent signal-to-noise-ratio and lack of confidence in the analysis.
• a porous surface effectively increases the number and density of possible DNA binding sites per unit area of the cross-section.
• the type of surface chemistry, ink composition, print pins size, and ink volume may effect sensitivity, though not light scattering.
• the distribution of the fluorescent molecules on the internal surfaces of the porous glass structure should overlap localized higher excitation intensity.
• Second, light emitted by the fluorescent molecules should be able to escape the porous structure to be observed and measured.
• the distribution of fluorescent molecules as a function of depth in the porous coating may have a dramatic effect on sensitivity.
• One can alter this distribution by modifying the density of binding sites or the number of molecules to be bound that are present in the ink. No matter what the concentration of biological material in printing inks one can achieve heightened sensitivity in the inventive porous substrates relative to conventional substrates.
• the samples are scanned for background auto-fluorescence after firing.
• the background fluorescence of the tinted porous glass is reduced compared to the original white porous layer by over 50% in terms of relative fluorescence units (RFU).
• REU relative fluorescence units
• the auto-fluorescent background on the tinted porous slides is comparable to the two-dimensional flat GAPS coated control slide, and the uncoated 1737-glass LCD panel.
• the porous slides were dip coated with a 5% GAPS solution to prepare them for attaching nucleotides.
• the GAPS coated porous slides were scanned again to compare the auto-fluorescent background due to the coating process.
• each porous slide was treated with a reducing agent, such as NaBH 4 which is used to decrease Cy3 background signal, and one of each slide were left untreated with the NaBH 4 .
• a reducing agent such as NaBH 4 which is used to decrease Cy3 background signal
• the microarrays were hybridized over night in a 42°C water bath. Afterwards, the hybridized slides were washed and scanned using a Genepix 4000B seamier for the analysis. A mean background was calculated. The background reflectance in the tinted porous had been reduced significantly compared to the original white porous glass.
• the results are represented in Figure 2, which indicate that the signal to noise ratio is lower in the tinted porous substrate with respect to the Cy5 signal.
• the tinted porous substrate exhibited less than about 20% or 25% background fluorescence relative to that shown for the "white" porous substrate.
• the tinted substrate displayed only about V ⁇ to about V 5 of the background of the white substrate.
• This phenomenon may be attributable to the advantage of a tinted porous glass substrate layer as well as in part, with respect to the Cy3 signal, to the NaBH 4 treatment reducing organic auto-fluorescence impurities due to the GAPS coating.
• Figures 3 A and 3B show, when compared to the original white porous glass, the net signal (Mean signal - Mean background) for both Cy3 and Cy5 fluorescence is lower in the tinted porous glass.
• Figures 4A-4C are images of three substrates after firing.
• Figure 4A presents the surface of a typical 1737-glass panel, which serves here as a control. Notice that the non-porous substrate exhibits relatively minor levels of auto-fluorescence as data in the foregoing graphs support.
• An example of the tinted porous glass of the present invention in Figure 4B stands rather favorably in comparison, to the non-porous flat glass surface in terms of relative ⁇ background signal.
• the "white" porous glass substrate of Figure 4C exhibits a relatively high degree of background fluorescence, as data in the aforementioned graphs confirm.
• the devices of the present invention help the performance of binding assays detect targets in samples.
• the target analytes preferably bind with probe molecule(s) immobilized on a surface of the substrate.
• the inventive device and method has been described in the context of nucleotide reactions for illustrative purposes, the present invention is not necessarily limited only to nucleotide hybridization assays. Alternate applications that may benefit from the present invention may include other biological binding-assay formats.
• the kinds of probe molecules which may be immobilized on a porous layer may be selected from a variety of biological or chemical species or molecules.
• the probe molecules may include proteins, peptides, polypeptides, protein-membranes, which may be useful for the emerging field of proteomics, G-coupled protein receptors, gangliosides, cells or cell membranes, cell-lysate, or protein-small molecule ligands, for drug compound molecule interactions.
• proteomics G-coupled protein receptors
• gangliosides cells or cell membranes
• cell-lysate cell-lysate
• protein-small molecule ligands for drug compound molecule interactions.
• the target analyte can be a nucleic acid.
• the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others.
• the target sequence is preferred used to be in a single-stranded format; however, the target sequence in a double stranded conformation (e.g., genomic DNA) may be used after denaturation.
• the target sequence is preferably labeled with a detectable moiety or moieties, such as fluorescence dye molecule(s) to allow detection of the binding of the target sequence to the probe microspots directly using fluorescence imaging techniques, or with biotin moieties in which a sequential detection step using labeled anti-biotin or anti-biotin coated gold nanoparticle is required for detection the binding of the target sequence to the probe microspots (Bao et al. Anal. Chem. 2002, 74, 1792-1797).
• a "probe nucleic acid” or “probe sequence” refers to a nucleic acid sequence with known sequence or defined sequence.
• the probe nucleic acid is a cDNA, a ohgonucleotide with defined sequence, or a modified ohgonucleotide with defined sequence.
• a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 1993, 49,1925), peptide nucleic acid backbones and linkages (Egholm, J. Am. Chem. Soc. 1992, 114,1895); Nielsen, Nature, 1993,365,566). As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention.
• a pharmacological compound or ligand may be the target compound or ligand when using a probe protein microarray.
• a "target compound” or “target ligand” in this context refers to a chemical or biochemical or biological compound whose identity, abundance, or binding affinity and specificity is to be detected.
• the target compound can be synthetic, naturally occurring, or biological produced.
• the target compound may be a street drug of abuse, a pharmaceutical drug candidate, a chemical (an organic or inorganic compound, including ionic salt), a biochemical (e.g., synthetic lipids, oligosaccharides, peptides, amino acids, nucleotides, nucleosides, etc), or a biological (e.g., a naturally occurring lipids, a protein, an antigen, an antibody, a growth factor, etc.).
• the target compound may be an activator, an inhibitor, an effector, a binding partner, or an enzyme substrate of the probe protein(s).
• the target compound can be part of a selected or random compound library.
• probe protein or “probe polypeptide” refers to a polypeptide with a known sequence.
• the probe proteins may be obtained from natural sources or, optionally, be overexpressed using recombinant DNA methods.
• the probe proteins may be either purified using conventional approaches or un-purified (e.g., cell lysates).
• the probe protein includes, but not limited to, intracellular proteins, cell surface proteins, soluable proteins, toxin proteins, synthetic peptides, bioactive peptides, and protein domains.
• intracellular proteins include, but are not limited to: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, kinases, phosphoproteines, and mutator transposons, DNA or RNA associated proteins (for example, homeobox, HMG, PAX, histones, DNA repair, p53, RecA, robosomal proteins, etc.), electron transport proteins (e.g., flavodoxins); adaptor proteins; initiator caspases, effector caspases, inflammatory caspases, cyclins, cyclin-dependent kinases, cytokeletal proteins, G-protein regulators, small G proteins, mitochondria-associated proteins, PDZ adaptor proteins, PI-4-kinases, etc..
• Applicable cell surface proteins include, but are not limited to: G-protein coupled receptors (e.g., aderenergic receptor, angiotensin receptor, cholecystokinin receptor, muscarinic acetylcholine receptor, neurotensin receptor, galanin receptor, dopamine receptor, opioid receptor, erotonin receptor, somatostatin receptor, etc), G proteins, ion-channels (e.g., nicotinic acetylcholine receptor, sodium and potassium channels, etc), receptor tyrosine kinases (e.g., epidermal growth factor (EGF) receptor), immune receptors, integrins, and other membrane-bound proteins.
• G-protein coupled receptors e.g., aderenergic receptor, angiotensin receptor, cholecystokinin receptor, muscarinic acetylcholine receptor, neurotensin receptor, galanin receptor, dopamine receptor, opioid receptor,
• Toxin proteins include, but are not limited to, cholera toxin, tetanus toxin, sliiga toxin, heat-labile toxin, botulinum toxin A & E, delta toxin, pertussis toxin, etc. Toxin domains or subunits may also be used.
• the probe protein microarrays may be used to identify small molecule-binding proteins (Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlmgmaier, S., Houfek, T., et al. "Global analysis of protein activities using proteome chips” Science 2001, 293, 1201-2105), or used to measure protein kinase activities (Houseman, B.T., Huh, J.H., Kron, S.J., Mrksich, M.
• the target analyte may be an antigen, a hormone, a cytokine, an immune antibody, a protein, a lipid, or a mixture of un-purified cell lysate, when a probe antibody microarray is used.
• target biologicals herein means a biological from a biofluid or an organelle or a living cell whose identity/abundance is be detected.
• the probe antibody includes, but not limited to, an immunoglobulins (e.g, IgEs, IgGs and IgMs), a therapeutically or diagnostically relevant antibodies (e.g., antibodies to human albumin, apolipoproteins including apolipoprotein E, human chorionic gonadotropin, cortisol, a-fetoprotein, thyroxin, thyroid stimulating hormone, antithrombin; antibodeis to antieptileptic drugs (phenytoin, primidone, carbariezepin, ethosuximide, valproic acid, and phenobarbitol), cardioactive drugs (digoxin, lidocaine, procainamide, and disopyramide), bronchodilators (theophylline), antibiotics (chloramphenicol, sulfonamides), antidepressants, immunosuppresants, abused drugs (amphetamine, methamphetamine, cannabinoids, cocaine and opiates)), a
• cholerae Escherichia, e.g., Enterotoxigenic E. coli, Shigella, e.g., S. dysenteriae; Salmonella, e.g., S. typhi; Mycobacterium e.g., M. tuberculosis, M. leprae; Clostridium, e.g., C. botulinum, C. tetani, C. difficile, C.perfringens; Comyebacterium, e.g., C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g., S.
• aureus Haernophilus, e.g., H. influenzae; Neisseria, e.g., N. meningitidis, N. gonorrhoeae; Yersinia, e.g., Y. lamblia, Y. pestis; Pseudomonas, e.g., P. aeruginosa, P. putida; Chlamydia, e.g., C. trachomatis; Bordetella, e.g., B. pertussis; Treponema, e.g., T.
• Haernophilus e.g., H. influenzae
• Neisseria e.g., N. meningitidis, N. gonorrhoeae
• Yersinia e.g., Y. lamblia, Y. pestis
• Pseudomonas e.g.
• an antibody to bacteria toxin e.g., antibodies to diphtheria toxin, anthrax toxin, tetrodotoxin, saxitoxin, bactrachotoxin, grayanotoxin, veratridine, actonitine, scorpion, sea anemone venom, scorpion charybdotxins, dendrotoxins, hanatoxins, sea anemone toxins, hololena, calcicludine, bungarotoxin, cholera toxin, conantokin, etc).
• an antibody to bacteria toxin e.g., antibodies to diphtheria toxin, anthrax toxin, tetrodotoxin, saxitoxin, bactrachotoxin, grayanotoxin, veratridine, actonitine, scorpion, sea anemone venom, scorpion charybdotxins, dendrotoxins, hanatoxins, sea ane
• the probe antibody arrays may be used for protein profiling, measurement of protein abundance in blood, measurement of cytokine abundances, detection of bacteria toxins in samples (such as environmental water, or food resources), as well as capture of leukocytes/phenotyping leukemias.
• target species may be present in any number of different sample types, including, but not limited to, bodily fluids including blood, lymph, saliva, vaginal and anal secretions, urine, feces, perspiration and tears, and solid tissues, including liver, spleen, bone marrow, lung, muscle, brain, etc.
• the "probes" can also be antigens, in which the antigen arrays may be used for reverse immunoassay to measure immuno- antibodies and allergens.
• a carbohydrate microarray having oligosaccharides or polysaccharides immobilized on to a surface at defined locations may be used to detect carbohydrate-binding protein target(s) in a sample (Fukui, S., Feizi, T., Galustian, C, Lawson, A.M., and Chai, W.

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